Aim: To analyze the sustained release of intracanal medicaments with or without a carrier and testing their antimicrobial efficacy in root canal against Candida albicans and Enterococcus faecalis.Materials and Methods: A total of 80 single-rooted anterior teeth were selected, root canal preparation was done, and teeth were divided into two halves and contaminated with C. albicans and E. faecalis, which were further divided into four test groups each according to intracanal medicaments used. Chitosan was used as vehicle for triple antibiotic paste (TAP) and calcium hydroxide and antimicrobial assessment was performed on second and seventh day. Dentine samples were collected after eachtime intervals and the number of colony-forming units (CFUs) was determined.Results : All four medicaments used in this study showed antifungal and antibacterial activity. Group II (TAP + chitosan) and Group III (Ca(OH)2 + chitosan) had the higher antifungal and antibacterial activity compared with two other groups.Conclusion: Combination of TAP + chitosan and Ca(OH)2 + chitosan produced better results compared with the combination of medicaments with saline.

Elimination of microorganisms and complete removal of pulp tissue from the root canal system is of dominant magnitude during endodontic therapy. The root canal success mainly depends on mechanical preparation, irrigation, microbial control, and complete filling of the root canal system. Microorganisms, bacteria, and their products are considered as the etiological agents of pulp necrosis and periradicular lesions. They may survive during endodontic procedures due to anatomical structural complexities and limitations of access by instrumentation and irrigants. To ensure complete elimination of root canal bacteria, effective antimicrobial agents are required for a predetermined time period for predictable eradication of remaining bacteria. [1]

Candida albicans is the most common species of fungi cultured from root canals of teeth with failed endodontic treatment. Because of collagenolytic activity, it may be possible for the yeast to use dentin as a nutrient source and promote colonization in the root canal. [2] Another organism commonly found in cases of failed endodontic infections and endodontic flare-ups is Enterococcus faecalis. It has the ability to survive in root canal system as a single organism without the support of other bacteria and is small enough to proficiently invade and live within the dentinal tubules. [3]

Many studies have reported that C. albicans and E. faecalis are able to invade dentinal tubules to variable depth. So, to ensure complete elimination of root canal bacteria, an effective antimicrobial agent in the root canal is required for a predetermined time period for complete eradication of any remaining bacteria. Calcium hydroxide has been widely used as an intracanal medicament in endodontics, and it has been demonstrated that C. albicans and E. faecalis have been reported to be resistant to the antimicrobial effect of calcium hydroxide. [4]

Triple antibiotic paste (TAP) is a mixture of metronidazole, ciprofloxacin, and minocycline. TAP has been used as an intracanal medicament for disinfecting the root canals during regenerative procedures. [5] The rationale of using TAP in this study is that the infected root canal is inaccessible to the local immune system and the concentration of the drug that reaches the canal space after administration of systemic antibiotics is minimal and unlikely to inhibit the bacterial growth. Therefore, the local application of antibiotics within the root canal system may be a more effective mode of delivering the drug. [6]

To increase the intracanal medicament stability, insolubility chitosan can be used as a drug carrier where it has added advantage of slow and controlled release of intracanal medicament. [4],[7],[8] Chitosan is produced by the partial deacetalylation of chitin. Chitin is the second most abundant natural polysaccharide composed of β (1→4) linked N-acetyl glucosamine units. Chitosan has a number of important pharmaceutical applications. It has been used in drug delivery, has an absorption enhancer, colon targeting and gene delivery. [9] Till date there is no sufficient data on the antimicrobial effect of intracanal medicaments using chitosan as a carrier on C. albicans and E. faecalis.

This study has compared the antimicrobial efficacy of TAP and calcium hydroxide with newer vehicle, i.e. chitosan, against C. albicans and E. faecalis. The aim of the present study is to analyze the sustained release of TAP and calcium hydroxide as intracanal medicaments in root canals using chitosan as a carrier and testing their antimicrobial efficacy against C. albicans and E. faecalis.

Materials and methods

0Sample preparation

A total of 80 freshly extracted single-rooted anterior teeth were selected for this study. Using rotary diamond disk, all the teeth were decoronated and standardized to 16 mm in length. The internal diameter of the root canal was prepared and standardized using Gates Glidden (GG) drill number 3 (Mani Inc., Japan). The organic and inorganic root canal preparation debris were removed by treating the teeth with 17% ethylene diamine tetra acetic acid (EDTA) for 5 min, followed by 5% sodium hypochlorite (Vishal Dentocare Private Limited, Ahmedabad, India) for 5 min.

Next teeth were dipped in distilled water for 5 min to remove any remaining remnants of irrigants and sterilized in an autoclave at 121°C for two cycles. In the second set of sterilization, teeth were divided into two halves. First half (n = 40) of the teeth were immersed in 1 ml of brain heart infusion (BHI) broth in individual microcentrifuge tubes. The other half (n = 40) of the teeth were immersed in Sabouraud's dextrose agar (SD) broth for the better penetration of the broth into the dentinal tubules. This methodology was similar to the one that was used by Krithikadatta et al. [10]

Contamination of the teeth

The test organisms chosen to use for this study were C. albicans and E. faecalis as they are the most frequently isolated bacterial species from the root canals of endodontically failed teeth. Twenty-four hour colonies of pure culture of C. albicans (American Type Culture Collection (ATCC) 2091) and E. faecalis (ATCC 35550) were grown on SD agar and BHI agar, respectively, and were suspended in 5 ml of SD broth for C. albicans and BHI broth for E. faecalis and incubated for 24 h at 37°C. Bacterial growth changes in turbidity were compared with 0.5 McFarland standard against a ruled paper, which can be comparable with a bacterial suspension of 1.5 × 10 8 CFU(colony-forming unit)/ml. A total of 50 μl of inocula were transferred to presterilized individual microcentrifuge tubes containing 1 ml of respective broths and teeth. All the procedures were carried out in a biosafety cabinet. The purity of the culture was checked by subculturing 5 μl of the broth from the incubated teeth in the respective broths, on agar plates. Contamination of the teeth was carried out for a period of 21 days.

Antimicrobial assessment

At the end of 21 st day, the teeth were irrigated with 5 ml of sterile saline to remove the incubation broth. In each group the teeth were assigned to the following groups (n = 5) for second and seventh day. A total of 40 teeth were used for C. albicans and 40 teeth for E. faecalis.

Intracanal medicaments were prepared for four groups:

Group I: TAP + saline

Group II: TAP + chitosan

Group III: calcium hydroxide [Ca (OH) 2 ] + chitosan

Group IV: Ca(OH) 2 + saline

All the teeth after medication were sealed with temporary restorative material and incubated in an aerobic environment at 37°C.

An antimicrobial assessment was performed at the end of 2 days and 7 days, with five teeth from each group, for each time interval. The teeth were washed with 5 ml of sterile saline to remove the medicament. Dentin debris was harvested at depth of 200 μm by using GG drills number 4 and collected in 1 ml of phosphate buffer saline solution. Diluted solution was transferred to their respective medium for culture. Plates were incubated for 24 h at 37°C. After 24 h, colonies of bacteria were counted using classical bacterial counting technique (Collins et al., 2004) and they were counted as number of CFU. [11] Number of CFU between second day and seventh day were compared for all the four groups.

Statistical analysis

The statistical package SPSS (Statistical package for social science, version 4) was used for statistical analysis. The data were analyzed with Wilcoxon-signed rank test to check the differences in microbial inhibition between the groups (P < 0.05). The Kruskal - Wallis analysis of variance (ANOVA) was used to check the differences in growth at different time intervals within the groups (P < 0.05). Mean and standard deviation were estimated for all the four different groups. [Table 1] shows the mean counts and standard deviation for CFUs between four different groups. Mean CFUs were compared for all the four groups. The inference from the table suggests that there was statistically significant difference among the groups (P < 0.001).

Table 1: Mean counts and standard deviation of CFU between four experimental groups

All the medicaments used in this study exerted antifungal activity against C. albicans. Ca(OH) 2 + chitosan and TAP + chitosan had the highest antifungal activity compared with TAP + saline and Ca(OH) 2 + saline. Ca(OH) 2 + chitosan has shown better antifungal efficacy against C. albicans compared with TAP + chitosan. For all the four groups, fungal inhibition was significantly more on the seventh day as compared with second day. There was no statistical significance seen at the depth of 200 μm.

For E. faecalis

All the medicaments used in this study exerted antibacterial efficacy against E. faecalis. Ca(OH) 2 + chitosan and TAP + chitosan had the highest antimicrobial activity compared with TAP + saline and Ca(OH) 2 + saline. For all the four groups, bacterial inhibition was significantly more on the seventh day as compared with second day. There was no statistical significance seen at the depth of 200 μm.

Discussion

To increase the efficiency of instrumentation, root canal irrigating solutions and intracanal medicaments are used to eliminate the bacteria from the root canals. [12] Antibiotics are used in dentistry both systemically and topically. During systemic administration of antibiotics, negligible concentrations reach the root canal, whereas during the local administration of antibiotics, greater concentrations can be used as intracanal medicaments, to decrease systemic consequences and complications. Because of the complexity of root canal infection, single irrigant or a medicament or an antibiotic could not result in effective sterilization of the root canal. Combination of irrigants or medicaments decreases the development of resistant bacterial strains and produces synergistic effect, whose antimicrobial action lasts longer and also sustains release of medicaments occurs. [13]

Chitosan is a natural polysaccharide comprising copolymers of glucosamine and N-acetyl glucosamine. Chitosan is used for preparation of nanoparticles of various applications because of its biodegradable and nontoxic properties. It is insoluble in acidic conditions and free amino groups on its polymeric chain protonates and contributes to its positive changes. Mechanism of action of chitosan is thought to be that cationically charged amino group may combine with anionic components such as N-acetyl muramic acid, sialic acid, and neuramic acid on the cell surface and suppresses growth of bacteria by impairing the exchanges with medium, chelating transition metal ions, and inhibiting enzymes. [8] Therefore, chitosan has been added to TAP and Ca(OH) 2 in an attempt to test the potential additive or synergistic effect on the viability of C. albicans and E. faecalis.

Ca(OH) 2 has limited ability to eradicate bacterial cells inside dentinal tubules. Ca(OH) 2 has low solubility, diffusibility, and dentin-buffering ability, which reduced its antibacterial effect. When Ca(OH) 2 is used alone without any combination with other antimicrobial agents, there is limited antimicrobial activity against E. faecalis. [14] This could explain that there occurs limited antimicrobial effect of Ca(OH) 2 alone when compared with Ca(OH) 2 combined with chitosan against C. albicans and E. faecalis.

The possible reason for the antimicrobial action of Ca(OH) 2 + chitosan might be due to the mechanism of action of chitosan that possesses the positively charged NH3 + groups of glucosamine that interacts with negatively charged surface components of bacteria, resulting in extensive cell surface attraction, leakage of intracellular substances, and causing damage to vital bacterial activities. [8] It may be possible that Ca(OH) 2 combined with chitosan inhibit the growth of C. albicans and affects their re-entry and recolonization. A synergistic/additive effect was found in Ca(OH) 2 combined with chitosan against C. albicans and E. faecalis when compared with Ca(OH) 2 combined with saline.

In a study conducted by Shaymaa et al., Ca(OH) 2 combined with chitosan solutions were more effective in inhibiting the growth of E. faecalis when compared with Ca(OH) 2 mixed with saline. [15] Ballal et al., in 2010 concluded that addition of chitosan to Ca(OH) 2 paste as an intracanal medication has been shown to promote prolonged calcium ion release. [16] In the present study Ca(OH) 2 + chitosan combination was more effective in inhibiting the growth of E. faecalis and C. albicans when compared with Ca(OH) 2 + saline combination. Ballal et al., reported that 2% chlorhexidine (CHX) gel combined with chitosan has shown highest antimicrobial effect against C. albicans and E. faecalis when compared with CHX gel or 2% chitosan alone. [4]

The use of the three Mix-MP TAP was developed by Hoshino and colleagues. It consists of ciprofloxacin, metronidazole, and minocycline. It is effective for disinfection of the infected necrotic tooth, setting conditions for the subsequent revascularization of the tooth. In an experimental study on dogs, the intracanal drug delivery of a 20 mg/ml solution of TAP resulted in >99% reduction in mean CFU levels. [17] TAP + chitosan showed better antimicrobial efficacy against C. albicans and E. faecalis. The probable reason might be chitosan as a drug carrier has the advantages of slow and controlled drug release, which improves drug solubility, stability, enhancing efficacy, and reduced toxicity. [8] Jadhav et al., in their case series when equal proportions of the components of TAP were ground and mixed with distilled water to a thick paste consistency resulted in successful revascularization of immature maxillary anterior teeth. [18] Alireza Adl et al., reported that TAP + saline combination has shown better antimicrobial effect against E. faecalis when compared with Ca(OH) 2 + saline combination. Ca(OH) 2 + saline combination was effective at a concentration of 200 μg/ml against E. faecalis. [19] Aleksandra et al., in their study found that chitosan has shown better antifungal activity against C. albicans. [20] In the present study, TAP + chitosan combination has shown better antibacterial and antifungal activity against C. albicans and E. faecalis when compared with TAP + saline combination.

Krithikadatta et al., found that the overall percentage inhibition of bacterial growth was 100% with 2% CHX gel when compared with 2% metronidazole gel, bioactive glass, and Ca(OH) 2 . [10] Hoshino et al., investigated the antibacterial effect of a mixture of ciprofloxacin, metronidazole, and minocycline with and without the addition of rifampicin on bacteria from infected root canals and concluded that alone none of the drugs resulted in complete elimination of bacteria. However, in combination these drugs were able to consistently sterilize all samples. In vitro, TAP was effective in removing commonly isolated endodontic pathogens from infected root canals. [21] Many in vitro studies have also reported that TAP was an effective disinfectant. [22]

Aggarwal et al., in their case compared the regenerative potential of Ca(OH) 2 and TAP in immature permanent teeth and concluded that critical step in regenerative therapy is complete disinfection of the root canal space using copious irrigation, minimal instrumentation, and placement of antibiotic paste. Ca(OH) 2 could weaken the root dentin and also because of its high pH could destroy vital apical cells including stem cells of apical papilla (SCAP). [23] Bose et al., reported that when Ca(OH) 2 and TAP were used as intracanal medicaments in immature necrotic teeth, they showed development of pulp dentine complex. [24] Sato et al., reported that TAP destroys bacteria in deep areas of root canals. [25] Sonali et al., in their study found that there was nonsurgical healing of large periapical lesions when TAP was used as intracanal medicament. [5] The findings of this study demonstrated that TAP + chitosan combination has shown higher antifungal and antibacterial activity against C. albicans and E. faecalis. However, chitosan alone may not be very useful as an antimicrobial agent against C. albicans and E. faecalis.

Further studies are required to use these medicament combinations with chitosan as a drug carrier in in vivo studies. Great care has to be taken when administering antibiotics locally and also patient may have sensitivity to chemicals or antibiotics. The depth of penetration of the medicament combinations into dentinal tubules, their duration of action, concentration of the medicaments, and volume of the medicament to be given are to be investigated and compared. However, preclinical and clinical trials are required for the evaluation of biocompatibility and safety to recommend these intracanal medicament combinations for clinical usage.

Conclusion

Combination of TAP + chitosan and Ca(OH) 2 + chitosan produced better results compared with the combination of medicaments with saline. Findings of this study demonstrated that combining TAP and Ca(OH) 2 with chitosan as a carrier had a good antimicrobial effect against C. albicans and E. faecalis.